This project seeks to design inhibitors of neural proteases associated with causative events in Alzheimer's Disease (AD), and has as its ultimate goal, the development of effective clinical drug candidates for AD therapy. The emerging molecular picture of AD suggests a complex, multi-factor etiology, however, mounting evidence implicates abnormal proteolytic processing of amyloid precursor protein (APP) as a key defect. Defining the exact nature of abnormal protease involvement in AD, including APP catabolism and perhaps, NGF receptor truncation, and developing inhibitors to block protease-mediated pathogenic events leading to Ad are the focal points of this project. The specific aims of the project include: 1) development of fluorogenic substrate probe molecules to assist in identifying aberrant Ad- related protease activity that mediates beta-amyloid formation or NGF receptor truncation; 2) design and synthesis of protease inhibitor probes for structural and biophysical studies of target proteases; 3) development of strategies for multiple fluorogenic substrate synthesis and screening protocols to assess protease substrate sub-site specificities; 4) comparative homology modeling of target protease three-dimensional structure; 5) experimental determination of target protease three- dimensional structure by X-ray crystallography and 2D, 3D and 4D NMR; 6) experimental determination of the three-dimensional structures of target protease-inhibitor complexes by X-ray crystallography and 2D, 3D and 4D NMR, to enable structure-based drug design, and 7) development of selective inhibitors of AD-related proteases based on structural and biophysical studies, and on computer-assisted drug design concepts. In order to achieve these objectives, a broad spectrum of molecular techniques will be deployed. Synthetic chemistry will generate specially tailored probes to identify AD proteases and assist in elucidation of the pathogenic role of target proteases. Other molecular probes that interact in a specific manner with target proteases will be used to augment structural and biophysical studies. Large arrays of fluorogenic substrate probes will be developed to provide substrate sub-site specificity data that will enable first generation, selective protease inhibitors to be designed. X-ray crystallography and NMR studies will determine the three-dimensional structure of target AD proteases, and the structures of protease-inhibitor complexes will be solved to enable structure-based drug design. An iterative process of inhibitor design and synthesis, complex structure determination, and biological testing will lead to inhibitor candidates that will serve as lead structures for ultimate medicinal chemistry refinement and clinical drug candidate development.